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Mesenchymal stem cells capable of continuously over-expressing IFN-gamma and application thereof

A quality stem cell, overexpression technology, applied in the field of stem cells, can solve the problem of lack of prevention of aGVHD, and achieve the effect of reducing the incidence or severity, high expression efficiency and high efficiency

Pending Publication Date: 2020-02-21
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In summary, there is no effective method for preventing aGVHD in the prior art. The present invention intends to provide a human umbilical cord mesenchymal stem cell that continuously overexpresses IFN-γ, so as to prevent the occurrence of aGVHD or reduce its severity without affecting GVL effect

Method used

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  • Mesenchymal stem cells capable of continuously over-expressing IFN-gamma and application thereof
  • Mesenchymal stem cells capable of continuously over-expressing IFN-gamma and application thereof
  • Mesenchymal stem cells capable of continuously over-expressing IFN-gamma and application thereof

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preparation example Construction

[0035] The present invention also provides a method for preparing the above-mentioned mesenchymal stem cells continuously overexpressing IFN-γ, comprising the following steps:

[0036] A lentiviral vector expressing IFN-γ was constructed and the viral particles were packaged; the frozen third-generation human umbilical cord mesenchymal stem cells were recovered, and after medium replacement, culture, and passage, the fifth-generation human umbilical cord mesenchymal stem cells in the logarithmic growth phase were selected. Mesenchymal stem cells, when the cells grow to 80-90% confluence, digest with trypsin and count, 37°C, 5% CO 2 Cultivate overnight under conditions; virus particles infect cells, 37°C, 5% CO 2 Change the medium after 24 hours of cultivation under the conditions, and replace with the screening medium after 48 hours, 37 ° C, 5% CO 2 Under these conditions, the recombinant gene-modified mesenchymal stem cells can be obtained.

[0037] Further, the selection m...

Embodiment 1

[0041] Example 1 Construction of IFN-γ overexpression vector

[0042] (1) Select the target gene: the cDNA sequence of IFN-γ is determined by Pubmed search. The nucleotide sequence of the target gene is shown in SEQ ID NO:1.

[0043] The primers of the target gene are shown in SEQ ID NO:2 and SEQ ID NO:3.

[0044] Upstream primer: 5'-CGGAATTCATGAAATATACAAGTTAT-3'. (SEQ ID NO:2)

[0045] Downstream primer: 5'-CGGGATCCTTACTGGGATGCTCTTCGA-3'. (SEQ ID NO:3)

[0046] The restriction enzymes are: EcoR I and BamH I.

[0047] (2) Ligation of target gene and shuttle vector and result analysis and identification:

[0048] The target gene was amplified by PCR from the plasmid, and the pCDH vector and IFN-γ gene were digested with two restriction enzymes EcoR I and BamH I; 4 The ligase respectively connects the IFN-γ gene and the pCDH vector fragment to construct the recombinant plasmid pCDH-IFN-γ.

[0049] The basic process of target gene amplification is as follows:

[0050] ①T...

Embodiment 2

[0077] Example 2 Construction of recombinant lentiviral vector

[0078] The preparation principle and process are as follows:

[0079] The lentiviral packaging system is a three-plasmid system, the auxiliary plasmids are pSPAX2 and pMD2G (from the State Key Laboratory of Biotherapy of Sichuan University), and the screening marker is puromycin.

[0080] (1) Plasmid preparation:

[0081] Plasmid preparation was performed using TIANGEN endotoxin-free plasmid extraction kit (Tiangen Biochemical Technology Co., Ltd., Beijing).

[0082] The preparation steps of the plasmid were carried out in detail according to the instructions of Tiangen Endotoxin-Free Plasmid Extraction Kit DP-117.

[0083] (2) Transfection

[0084] ① 24 hours before transfection, digest 293T cells in the logarithmic growth phase with trypsin, adjust the cell concentration to 6×10*5 / ml with DMEM medium containing 10% fetal bovine serum, re-seek in a 10 cm culture dish, and place at 37 ℃ with 5% CO 2 When the...

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Abstract

The invention belongs to the technical field of stem cells, and particularly relates to mesenchymal stem cells capable of continuously over-expressing IFN-gamma and application thereof. Aiming at theproblem that an effective method for preventing aGVHD is lacked in the prior art, the invention provides the mesenchymal stem cells capable of continuously over-expressing IFN-gamma. A recombinant vector contained in the stem cells contains a gene capable of continuously encoding the IFN-gamma, and can continuously over-express the IFN-gamma. The preparation method of the mesenchymal stem cells capable of continuouslu over-expressing the IFN-gamma is simple; the stem cells simultaneously play anti-tumor and immune roles in an organism after allogeneic hematopoietic stem cells are transplanted,the occurrence rate or severity of aGVHD is reduced, meanwhile, the GVL effect is not influenced, and a new method is provided for prevention and treatment of the aGVHD after the allogeneic hematopoietic stem cells are transplanted. The expression vector disclosed by the invention is higher in expression efficiency, higher in infection efficiency and better in expression stability.

Description

technical field [0001] The invention belongs to the technical field of stem cells, and in particular relates to a mesenchymal stem cell continuously overexpressing IFN-γ and its application. Background technique [0002] The main feature of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the activation of immune cells such as T lymphocytes of the donor. Through the immune mechanism, the remaining leukemia cells of the recipient are eliminated to the greatest extent, in order to achieve graft resistance. Leukemia effect (graft-versus-leukemia, GVL). The activation of donor T cells in the recipient often causes acute graft-versus-host disease (aGVHD), which is the most serious complication of allo-HSCT and seriously affects the success rate of transplantation. Its main mechanism is that donor T cells act on recipient cells by activating antigen-presenting cells and inducing the release of cytokines, causing damage to target organs such as the patient's skin,...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12N15/23A61K35/28A61P37/06
CPCA61K35/28A61P37/06C07K14/57C12N5/0668C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 贾永前陈影影吴鹏强王甫珏
Owner SICHUAN UNIV
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